The present invention relates to a method of diagnosing certain leukemias. More specifically, it concerns a method of detecting an RNA sequence which represents a protein or proteins which are phenotypically characteristic of certain leukemias.
Approximately 88% of the patients with clinically typical chronic myelogenous leukemia (CML) have a cytogenetic abnormality known as the Philadelphia chromosome (Ph.sup.1). This aberration has also been reported at a much lower frequency in both acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML). Chromosome banding techniques have shown the Ph.sup.1 chromosome to be a shortened chromosome 22 with a break occurring in bands q11. In 92% of the patients with Ph.sup.1 -positive CML, the missing piece of chromosome 22 attaches distally to the long arms of chromosome 9, band q34, in what has been shown to be a reciprocal translocation. Of the Ph.sup.1 -positive variants with a different type of alteration, 4% are known to have complex translocations which again involve chromosome 9.
Specific karyotype abnormalities are also associated with other kinds of malignancies. For example, other types of human and murine leukemias and lymphomas have been correlated with particular cytogenetic changes. Additionally, it is known that certain leukemias with distinct cytogenetic changes display constitutive expression of proteins which are normally regulated during myeloid differentiation. Thus, phenotypic subcategories of leukemia, as defined by karyotype abnormalities, can additionally be defined by protein alterations which in turn may reflect the cellular populations of RNA.
Since preferred treatments differ for different malignancies, there is always a need for methods of diagnosis which are improvements in terms of speed, reliability, and/or cost. The present invention provides an improved diagnostic method by using genetic engineering techniques to detect genetic sequences coding for phenotypically characteristic proteins.
The genetic engineering techniques used relate to the recent advances in recombinant DNA technology which have facilitated the isolation of specific genes or parts thereof and their transfer to bacteria, yeast, plant, or animal cells and to the viruses that infect these organisms. The transferred gene material (or modified gene) is replicated and propagated as the transformed cell or viruses replicate.
The transfer and expression of genes or portions thereof between viruses, eukaryotes, and prokaryotes is possible because the DNA of all living organisms is composed of the same four nucleotides. The basic differences reside in the sequences in which the nucleotides appear in the genome of the organism. Specific nucleotide sequences, arranged in codons (nucleotide triplets), code for specific amino acid sequences. However, the coding relationship between an amino acid sequence and a DNA nucleotide sequence is essentially the same for all organisms.
Many recombinant DNS techniques employ transfer vectors. A transfer vector is a DNA molecular which contains genetic information which insures its own replication when transferred to a host microorganism strain. Plasmids are an example. "Plasmid" is the term applied to any autonomously replicating DNA unit which might be found in a microbial cell, other than the genome of the host cell itself. A plasmid is not genetically linked to the chromosome of the host cell. Plasmid DNA's exist as double stranded ring structures. A plasmid DNA ring may be opened and a fragment of heterologous DNA inserted and the ring reclosed. Thus, transfer vectors serve as a carrier or vector for an inserted fragment of heterologous DNA.
Transfer is accomplished by a process known as transformation. During transformation, host cells mixed with plasmid DNA incorporate into themselves entire plasmid molecules. Once a cell has incorporated a plasmid, the latter is replicated within the cell and the plasmid replicas are distributed to the daughter cells when the cell divides. Any genetic information contained in the nucleotide sequence of the plasmid DNA can be expressed in the host cell. Typically, a transformed host cell is recognized by its acquisition of traits carried on the plasmid, such as resistance to certain antibiotics. Any given plasmid may be made in quantity by growing a pure culture of cells containing the plasmid and isolating the plasmid DNA therefrom.
Restriction enzymes are also frequently used in these techniques. They are hydrolytic enzymes capable of catalyzing site-specific cleavage of DNA molecules. The locus of restriction enzyme action is determined by the existence of a specific nucleotide sequence. Such a sequence is termed the recognition site for the restriction enzyme. Since any DNA susceptible of cleavage by such an enzyme must contain the same recognition site, the same cohesive ends will be produced by the cleavage. Therefore, it is possible to join heterologous sequences of DNA which have been treated with a restriction endonuclease to other sequences similarly treated. Restriction sites are relatively rare, but the general utility of restriction endonucleases has been greatly amplified by the chemical synthesis of double stranded oligonucleotides bearing the restriction site sequence. Therefore, virtually any segment of DNA can be coupled to any other segments simply by attaching the appropriate restriction oligonucleotide to the ends of the molecule, and subjecting the product to the hydrolytic action of the appropriate restriction endonuclease, thereby producing the requisite cohesive ends.
Other methods for DNA cleavage or for end sequence modification are also available. A variety of nonspecific endonucleases may be used to cleave DNA randomly. End sequences may be modified by creation of oligonucleotide tails of dA on one end and dT at the other, or a dG and dC, to create sites for joining without the need for specific linker sequences.
The term "expression" is used in recognition of the fact that an organism ordinarily does not make use of all of its genetically endowed capabilities at any given time. Even in relatively simple organisms such as bacteria, many proteins which the cell is capable of synthesizing are not synthesized, although they may be synthesized under appropriate environmental conditions. When the RNA that codes for a given protein is being synthesized by the organism, that RNA is said to be expressed. The RNA synthesis will eventually lead to the synthesis of the corresponding protein.
The use of these techniques in the present invention is described below.